Development of an assay system for the analysis of host RISC activity in the presence of a potyvirus RNA silencing suppressor, HC-Pro

Background To investigate the mechanism of RNA silencing suppression, the genetic transformation of viral suppressors of RNA silencing (VSRs) in Arabidopsis integrates ectopic VSR expression at steady state, which overcomes the VSR variations caused by different virus infections or limitations of host range. Moreover, identifying the insertion of the transgenic VSR gene is necessary to establish a model transgenic plant for the functional study of VSR. Methods Developing an endogenous AGO1-based in vitro RNA-inducing silencing complex (RISC) assay prompts further investigation into VSR-mediated suppression. Three P1/HC-Pro plants from turnip mosaic virus (TuMV) (P1/HC-ProTu), zucchini yellow mosaic virus (ZYMV) (P1/HC-ProZy), and tobacco etch virus (TEV) (P1/HC-ProTe) were identified by T-DNA Finder and used as materials for investigations of the RISC cleavage efficiency. Results Our results indicated that the P1/HC-ProTu plant has slightly lower RISC activity than P1/HC-ProZy plants. In addition, the phenomena are consistent with those observed in TuMV-infected Arabidopsis plants, which implies that HC-ProTu could directly interfere with RISC activity. Conclusions In this study, we demonstrated the application of various plant materials in an in vitro RISC assay of VSR-mediated RNA silencing suppression. Supplementary Information The online version contains supplementary material available at 10.1186/s12985-022-01956-2.


Background
P1/HC-Pro fusion protein of potyvirus is the first identified viral suppressor of RNA silencing (VSR). The HC-Pro inhibits short-interfering RNA (siRNA) and microRNA (miRNA) regulatory pathways [1,2]. Several studies have demonstrated that P1 enhances the suppression ability of the HC-Pro [3,4]. Thus, the P1/HC-Pro fusion protein has been used to investigate the mechanism of silencing suppression [3]. The fusion protein can be cleaved by P1 protease activity in vivo to generate two separated P1 and HC-Pro proteins for adapting to potyvirus infection [5]. Several studies investigated the HC-Pro function through viral infection [6][7][8][9]. However, the unequal viral titers caused variation in HC-Pro amounts, which is challenging to evaluate the suppression efficiency. For instance, TuGK, a mild strain of turnip mosaic virus (TuMV) carrying the GFP gene, showed a lower viral titer than the severe strain TuGR (a wild-type TuMV carrying a GFP gene) [8]. Thus, the unequal viral titers between TuGK and TuGR result in different amounts of HC-Pros [8]. Moreover, different species viruses might have various host ranges, which limits the comparison of various HC-Pros on the same plant host. For instance, the zucchini yellow mosaic virus (ZYMV) cannot infect cruciferous plants. In contrast, TuMV cannot infect cucurbit species, which means the ZYMV HC-Pro (HC-Pro Zy ) and TuMV HC-Pro (HC-Pro Tu ) cannot be compared the suppression efficiency through the viral infection.
To overcome the titer variation and host range limitation, the ectopic expression of HC-Pros in various species using a transgenic plant approach is a good solution. Wu et al. ectopically expressed wild-type HC-Pro Zy in Arabidopsis (P1/HC-Pro Zy plant) to identify the critical amino acids on the FRNK motif of HC-Pro that are involved in VSR function [7]. Kung  , which exhibit comparable amounts of HC-Pro, and demonstrated that the Arg of the FRNK motif of HC-Pro Tu is critical for physical interaction with HEN1 (a methyltransferase for miRNA methylation) to result in HEN1 inhibition [8,10]. Therefore, approximately 50% of unmethylated miRNAs exist in the cytoplasmic of P1/HC-Pro Tu plants [10]. Surprisingly, Wei et al. demonstrated that inside of AGO1 from P1/HC-Pro Tu plants only contain methylated miRNAs [11]. The P1/HC-Pro Tu−K plant expressing an Arg182Lys mutation (FKNK) of HC-Pro (HC-Pro Tu−K ), resulting in a loss 40% of the suppression ability in RNA silencing [8]. Hu et al. also demonstrated that P1/HC-Pro Tu plants have lower AGO1 levels than transgenic Arabidopsis expressing the P1/HC-Pro gene of tobacco etch virus (TEV) (P1/ HC-Pro Te plant) and P1/HC-Pro Zy plants; however, three P1/HC-Pro plants exhibit a common severe serratedcurled leaf phenotype, suggesting that various HC-Pros exhibit different ability in suppressing RNA silencing [4].
Many studies imply that interfering with RNA-induced silencing complex (RISC) assembly triggers autophagy for AGO1 degradation [12][13][14]. To further apply genesilencing components or autophagy mutants to the other P1/HC-Pro plants, these T-DNA insertion positions should also be identified. T-DNA of P1/HC-Pro Tu gene was inserted in chromosome 1 (at the 28,814,559 th nt) [15], and the ATG8a (an autophagy gene) is located on chromosome 4. Wei et al. and Shang demonstrated ATG8a knockout mutant in a P1/HC-Pro Tu background (P1/HC-Pro Tu /atg8a ge plants) restores the AGO1 levels to the level near those found in Col-0 plants, which indicates that HC-Pro Tu triggers autophagic AGO1 degradation [11,16].
Investigating the RISC activity during TuMV infection is a novel undertaking. Many studies have demonstrated in vitro RISC activity assays using Flag-tagged AGO1 transgenic plant, which was generated in the ago1-36 background [17,18]. However, applying Flag-tagged AGO1 transgenic plants limits the RISC assay to plants with mutations in RNA silencing or these P1/HC-Pro plants. For instance, fifty-six different mutant alleles of AGO1 have been identified with exhibit losses of particular miRNA regulatory functions [19]. If we want to study particular functions for those ago1 mutants, we have to individually construct the Flag-tag on each mutated AGO1 gene and transfer them into the ago1-36 allele. Therefore, the best way is to generate an α-AGO1 antibody for endogenous AGO1 immunoprecipitation (AGO1-IP). Furthermore, Dicer-like 1 (DCL1) responds to miRNA biogenesis, whereas DCL2, DCL3, and DCL4 play roles in short-interfering RNA (siRNA) generation [20]. RNA-dependent-RNA polymerases (RDR2 and RDR6), HYPONASTIC LEAVES1 (HYL1), SERRATE (SE), and other AGOs (e.g., AGO2, AGO4, and AGO7), which are involved in small RNA regulatory pathways or virus defense. These RNA silencing components also have related mutant lines that can be used as plant materials for studies of their RISC activity [9,[21][22][23]. Therefore, an α-AGO1 antibody that specifically immunoprecipitates endogenous AGO1 from any Arabidopsis material could help researchers further investigate gene silencing.
In this study, we identified the T-DNA insertion in various P1/HC-Pro plants to provide genetic information that allows us to generate more plant materials for the in vitro RISC assay. We also developed an α-AGO1 antibody for the in vitro RISC assay to evaluate RISC activity in these P1/HC-Pro and virus-infected plants. We demonstrated that various HC-Pros of viral species have different RISC inhibition abilities, which opens a new field for further investigation of RNA silencing and suppression.

Endogenous AGO1 IP for the in vitro RISC assay
By using endogenous AGO1, the in vitro RISC assay can eliminate the limitation of plant material; however, a good-quality α-AGO1 antibody that does not affect AGO1 function would determine whether the in vitro RISC assay can be successfully performed. Although commercial AGO1 is available, we made a homemade α-AGO1 antibody to pull down a large amount of AGO1 for the assay. Moreover, the generation knowledge can be applied to producing other AGO antibodies, e.g., AGO2.
The amino acid alignments of AGO1, AGO2, AGO4, and AGO10 showed that the 1-to-256-aa region is variable, with 12.2%-30.5% identity (Fig. 1a). Thus, the N-terminus of AGO1 (1-240 aa) was selected for recombinant protein production and purified as an antigen to the AGO1 antibody (Fig. 1a, box). Western blot analysis showed that the α-AGO1 antibody could recognize endogenous AGO1 (117 kDa) in Col-0 plants. In contrast, the AGO1 signal was absent in the ago1-36 null mutant, suggesting that the homemade α-AGO1 antibody can be tested for its IP ability with an in vitro RISC assay (Fig. 1b).

Evaluating endogenous RISC activity in P1/HC-Pro plants
Our previous study generated three viral species of P1/ HC-Pro plants, including P1/HC-Pro Tu , P1/HC-Pro Zy , P1/HC-Pro Te , and P1/HC-Pro Tu−K plants (Fig. 3a) [4,8]. The P1/HC-Pro Tu , P1/HC-Pro Zy , and P1/HC-Pro Te plants showed identical elliptical cotyledons (Fig. 3a, arrowheads). However, the cotyledons of P1/HC-Pro Tu−K plants were round in shape similar to those of Col-0 (Fig. 3a). We applied NGS to obtain the genomic sequences from these P1/HC-Pro plants. We also developed T-DNA Finder software to identify the T-DNA insertion based on the genomic sequence profiles. Two T-DNAs were identified on chromosome 1: P1/HC-Pro Te (at the 16,780,881 th nt) and P1/HC-Pro Tu (at the 28,814,559 th nt) (Fig. 3b). In contrast, P1/HC-Pro Tu−K was inserted on chromosome 3 (at the 21,554,188 th nt), whereas P1/HC-Pro Zy was inserted on chromosome 4 (at the 10,075,981 th nt) (Fig. 3b). According to the TAIR database, several critical RNA silencing component genes, e.g., AGOs, DCLs, RDRs, HEN1, HYL1, SE, and ATG8a, were also labeled on diagrammatic chromosomes with these T-DNA insertions, which is helpful for the generation of relative mutations in these P1/HC-Pro plants by crossing or gene editing (Fig. 3b).
We performed western blotting to evaluate the endogenous AGO1 levels in various transgenic plants, including the L1 line and P1/HC-Pro Tu plants (Fig. 3c, panel i). Notably, the L1 line is a β-glucuronidase (GUS)-transgenic Arabidopsis plant that underwent sense-posttranscriptional gene silencing, and the T-DNA inserted on chromosome 3 (at the 2,350,388th nt) (Fig. 3b) [25]. The transgene of P1/HC-Pro Tu was transferred into the L1 line to generate P1/HC-Pro Tu plant [8]. The Col-0 and L1 lines showed identical endogenous AGO1 amounts, whereas P1/HC-Pro Tu plants showed lower levels of AGO1 while HC-Pro was presented (Fig. 3c, panel i) RUBISCO (asterisk) and tubulin were used as loading controls. d Immunoprecipitation for evaluating the interactions of AGO1 with various HC-Pros. The asterisks indicate the heavy chain of IgG. Tubulin was used as a loading control 0.7-to 0.9-fold of those found in the wild-type plants, suggesting that various species of HC-Pros have different AGO1 degradation efficiency (Fig. 3c, panel ii) [4,11]. Therefore, we aimed to understand whether endogenous AGO1 activity could be affected by VSR through in vitro RISC evaluation.

Evaluation of the AGO1-HC-Pro interaction
A conserved WG pair, which localizes near the FRNK motif, is identified in 113 potyviruses [26]. Pollari et al. demonstrated that HC-Pro of potato virus A (HC-Pro PVA ) physically interacts with AGO1 through transient expression with a cross-linked purification approach [26]. However, we performed AGO1-IP from various P1/HC-Pro plants with the α-AGO1 antibody. The IP results indicated that AGO1 could be detected in three P1/HC-Pro and Col-0 plants at the input and AGO1-IP samples (Fig. 3d). The immunoassay failed to detect HC-Pro in AGO1-IP samples from three P1/HC-Pro plants, suggesting that HC-Pro and AGO1 might not have a direct interaction but might exist a bridge protein to mediate an indirect interaction between HC-Pro and AGO1 (Fig. 3d).

HC-Pro-mediated RISC inhibition assay
We purified comparable amounts of AGO1-IP from the various transgenic plants for in vitro RISC assays to evaluate the status of AGO1 activity with MYB33-230 RNA substrates. The AGO1-IP from Col-0 seedlings exhibited 20% and 29% normalized cleavage efficiency in 10 ng and 20 ng AGO1-IP (Fig. 4a, panel i; and 4b).
To evaluate other HC-Pros inhibit RISC activity, the P1/HC-Pro Zy and P1/HC-Pro Te plants were used to perform their RISC activity to compare with P1/HC-Pro Tu and Col-0 plants (Fig. 4c). The relative RISC cleavage efficiency of P1/HC-Pro Zy plants showed no difference from that of Col-0 but differed from that of P1/  (Fig. 4c and 4d, panel i). Notably, the P1/HC-Pro Tu−K sample did not inhibit RISC activity, suggesting FKNK mutation lost HC-Pro Tu -specific RISC inhibition ability ( Fig. 4c and 4d, panel ii).

TuMV-infected Arabidopsis triggers endogenous AGO1 degradation
We subsequently evaluated the endogenous AGO1 in TuMV-infected Col-0 plants (Fig. 5a). TuGR infection caused symptoms with mosaic and up-curling systemic leaves, whereas TuGK-infected plants showed symptomless (Fig. 5a). Similar to P1/HC-Pro Tu plants, TuGR-infected Col-0 plants showed lower amounts of endogenous AGO1 than mock or TuGK-infected Col-0 plants (Fig. 5b). High abundances of HC-Pro and CP were detected in TuGR-infected plants, whereas lower amounts of HC-Pro and CP were detected in TuGKinfected Col-0 plants, which demonstrated uneven HC-Pro Tu and HC-Pro Tu−K amounts in infected tissues. Similarly, miRNA regulation suppression was also observed in TuGR-infected Col-0 plants. More than twofold higher levels of AGO1 (miR168), ARF16 (miR160), and MYB33 (miR159) were also detected in TuGR-infected Col-0 plants (Fig. 5c). In contrast, TuGK infection induces ARF16 accumulation to approximately 1.5-fold levels, whereas the RNA levels for AGO1 and MYB33 were similar to those obtained with the mock plants (Fig. 5c). These data indicated that miRNA regulation in TuGR-infected plants was also suppressed.

TuMV infection inhibits the RISC activity
We subsequently evaluated whether RISC activity might be affected during TuMV infection (Fig. 6a). We collected compatible amounts of AGO1-IP from mock, TuGR-, and TuGK-infected plants (Fig. 6b). The RISC assay demonstrated that the MYB33-230 substrate in TuGRinfected plants has lower cleavage efficiency compared with mock or TuGK-infected plants (Fig. 6c). As shown in Fig. 6c, 20 ng AGO1-IP from mock plants had 50.7% RISC activity, whereas 20 ng AGO1-IP from TuGR-and TuGK-infected plants had 38.5% and 43.9% RISC activity, respectively (Fig. 6a and c). These data demonstrated that severe TuMV infection inhibits RISC activity, which is consistent with the RISC results obtained in P1/HC-Pro Tu plants.

Discussion
Gain-of-function and loss-of-function genetic studies are fundamental approaches to understanding gene function. Agrobacterium-mediated transformation is popular for creating ectopic transgenic plants or T-DNA insertion mutants. However, a few studies have identified the T-DNA insertion position. Because NGS is more efficient and economical, identifying franking sequences of T-DNA insertion becomes achievable and with several benefits, including whether the insertion is on a gene body and which chromosome is inserted. In addition, transgenic plants with a transparent genetic background can introduce another mutation by crossing or CRISPR-Cas approaches. The T-DNA of P1/HC-Pro Tu plants is inserted on chromosome 1 without breaking any gene body; thus, the atg8a mutant that localizes in chromosome 4 can be introduced into the transgenic plant to generate the P1/HC-Pro Tu /atg8a ge plants [11,16]. We also identified the other T-DNA insertions in these P1/ HC-Pro plants. The insertion data with the locations of the RNA silencing component provide information for Fig. 6 Evaluation of the in vitro RISC cleavage efficiency using TuMV-infected plants. a MYB33-230 RNA substrates were cleaved by AGO1-IP from mock, TuGR-, and TuGK-infected Col-0 plants. The numbers indicate the amounts of AGO1-IP that were used for cleavage. b The AGO1-IP amounts from mock, TuGR-, and TuGK-infected Col-0 plants were evaluated by western blotting with α AGO1 IgG to normalize the RISC cleavage efficiency. In: input; IP: immunoprecipitation; FT: flow through. c Normalized RISC cleavage efficiency of AGO1-IP from mock, TuGR-, and TuGK-infected Col-0 plants crossing strategy decisions. For instance, the T-DNA of the P1/HC-Pro Zy plant is inserted on chromosome 4, which cannot introduce the atg8a mutant into this transgenic plant by crossing, and this deficiency might be overcome by gene editing. However, for introducing the other silencing mutants shown in Fig. 3b, [17]. This system should be conducted in the ago1-36 background to eliminate the impact of endogenous AGO1. Hence, establishing the Flag-AGO1 approach might be time-consuming for genetic screening and limit the utility of in vitro RISC assay to other mutants or transgenic plants when the simultaneous introduction of two elements, including the Flag-AGO1 transgene and ago1-36 locus, is needed. Moreover, the T-DNA insertion of Flag-AGO1 is unclear and might have a genetic linkage with the other mutants. T-DNA Finder can obtain the insertion information for Flag-AGO1/ago1-36 plants. In contrast, the polyclonal α-AGO1 antibody targets the 240 aa N-terminus of AGO1 and specifically immunoprecipitates endogenous AGO1. We can rapidly take advantage of the in vitro RISC assay to verify the cleavage efficiency in any mutant and plant. AGO1 has 56 different mutant alleles, and some alleles might lose certain functions [19]. For instance, ago1-42 is mutated in the PAZ domain, which might affect the miRNA loading; whereas the ago1-27 mutant might lose miRNA duplex binding [13,24]. The AGO1-IP from these mutants can be used to perform small RNA profiling, RISC activity, and the RISC complex assembly. In contrast, viral-infected Arabidopsis can also be applied to investigate various VSRs in RISC inhibition efficiency. Virus infection of various ago1 mutants can be used to investigate the interaction between VSRs and AGO1 through AGO1-IP with an α-AGO1 antibody.
Moreover, we demonstrated that the structure of the RNA substrate is critical for the in vitro RISC assay. The assay with the substrates ARF10-230 and ARF10-191 showed different cleavage efficiencies. We hypothesized that a targeting site length of the 3'-end might be essential. A 3'-end target site that is too long might form a complicated secondary structure, which may be difficult for AGO1 to access, as in the case of the ARF10-230 substrate. Thus, the RNA structure needs to be considered before determining the substrate sequence. We observed that miR159 and miR398 have more cleavage efficiency than miR164 and miR160. The amounts of miRNAs incorporated into AGO1 might cause a difference in the cleavage efficiency. The application of AGO1-IP with small RNA NGS can identify the AGO1-containing miRNA and siRNA profiles, which allows us to answer this question. Furthermore, AGO-IP from virus-infected plants can also obtain the viral siRNA profile, which can be applied in viral cleavage evaluation. AGO2 has been demonstrated to be involved in resistance to viral infection [27]. Therefore, we can apply the same approach to generate AGO2-specific antibodies for AGO2-IP and in vitro RISC assays in comparative studies.
Pollari et al. demonstrated that a WG pair of HC-Pro PVA plays a role for AGO1 subcellular colocalization and interaction [26]. Wei et al. also demonstrated that HC-Pro Tu colocalized with AGO1 but had no direct interaction, as revealed by Förster resonance energy transfer (FRET) analysis [11]. Indeed, the IP results from three P1/HC-Pro plants did not show that three HC-Pros directly interact with AGO1. However, Pollari et al. used a cross-linking approach to demonstrate the interaction of HC-Pro PVA coupled with AGO1 [26]. It has been proposed that HC-Pro Tu accelerates the aggregation of AGO1 and the host proteins to form RNA granules, which would recruit the colocalization of AGO1 and HC-Pro to cytoplasmic foci [28]. To summarize, we hypothesized that HC-Pro might interact with bridge host factors, which could also interact with AGO1, through the WG pair of HC-Pro, suggesting that HC-Pro and AGO1 might have an indirect interaction.
Because comparable AGO1-IP amounts from three P1/HC-Pro plants, the more attenuated RISC activity of the P1/HC-Pro Tu sample might not cause by decreased AGO1 levels. Wei et al. demonstrated that HC-Pro Tu specifically inhibits HEN1 activity, resulting in 50% unmethylated miRNAs that could not be loaded into AGO1 (Fig. 7a) [11]. However, HC-Pro Zy and HC-Pro Te did not inhibit HEN1, which might allow more methylated miRNAs to be loaded into AGO1 for target RNA cleavage (Fig. 7b). We hypothesized that reduced miRNA content, e.g., 50% methylated miR159, in AGO1-IP of P1/ HC-Pro Tu plants resulted in lower RISC cleavage efficiency (Fig. 7a). Although P1/HC-Pro Tu /atg8a ge plants still contain approximately 50% unmethylated miRNAs [11]; however, restoring AGO1 in the plants allows them to carry more methylated miRNAs to restore the RISC activity ( Fig. 4a and b). Moreover, P1/HC-Pro Te plants containing 100% methylated miRNAs showed less RISC activity, suggesting that multiple RISC interference mechanisms might exist in different potyviruses. In summary, the reduced AGO1 amount and less methylated miRNAs in AGO1-IP are the unique abilities of HC-Pro Tu to inhibit RISC activity, which means that TuMV can inhibit RISC activity more significantly compared with Col-0 and ZYMV-infected plants. In contrast, TEV infection may inhibit RISC less significantly than TuMV (Fig. 7).
Shiboleth et al. demonstrated that the FINK motif mutation of HC-Pro Zy still retained vigorous RNA silencing suppression, suggesting small RNA sequestering is not the only way for HC-Pro Zy inhibition RNA silencing but might exist multiple suppression approaches [6]. Wei et al. also demonstrated that HC-Pro Zy and HC-Pro Te still can slightly trigger AGO1 degradation [11]. Indeed, HC-Pro Tu did not have a small RNA binding ability but can enhance autophagic AGO1 degradation and RISC inhibition more than HC-Pro Zy and HC-Pro Te because of HEN1 inhibition [11]. Therefore, we proposed that conserved and diverse VSR functions exist simultaneously in different viral HC-Pros.

Conclusion
In this study, we demonstrated that T-DNA insertion information could be used to create more plant materials for experimental variations. The endogenous AGO1based in vitro RISC assay can easily be performed with these materials for RNA silencing studies. Furthermore, our data imply that various HC-Pros of potyviruses exhibit different RISC inhibition and AGO1 degradation abilities in gene silencing suppression.

Plant materials and virus inoculum
Arabidopsis thaliana Columbia ecotype (Col-0), P1/HC-Pro Tu , P1/HC-Pro Tu−K , P1/HC-Pro Zy , P1/HC-Pro Te , P1/ HC-Pro Tu /atg8a ge plants, ago1-27, and ago1-36 mutants were used in this study [7,8,11,16,24]. The ago1-27 mutant is an Ala992Val mutation at the C-terminus of Fig. 7 The working hypothesis for various HC-Pros in inhibiting the RISC activity. a The model of RISC inhibition by HC-Pro Tu . b The model to explain no effect on RISC inhibition by HC-Pro Zy and HC-Pro Te . The dark red lines represent as mature miR159. The blue lines represent other small RNAs (sRNAs). The gray lines indicate the miRNA*. The dark red color indicates the methylated miRNAs. The pink color indicates the unmethylated miRNAs. Me, the methylated miRNA AGO1, which is a weak allele mutant [24]. The ago1-36 mutant (salk_087076) is an AGO1 null mutant with a T-DNA insertion at the end of the PAZ domain [17]. Arabidopsis seeds were surface sterilized and vernalized at 4 °C for 1 day before being sown on Murashige and Skoog medium. The wild-type TuGR, and the mild strain TuGK were maintained on Nicotiana benthamiana as inocula. The 2.5-week-old Arabidopsis seedlings were mechanically inoculated with viral inoculum prepared from 0.5 g of infected N. benthamiana leaves using 2 mL of 0.01 M potassium phosphate buffer (pH 7.2). The symptoms or subsequent analysis were observed 12 days post-inoculation (dpi).

Antibody production
To produce the recombinant protein, the pET28a-AGO1-N plasmid was cloned and transformed into the E. coli BL21 system. The recombinant proteins were expressed in 500 mL LB culture and then induced with 0.5 mM IPTG at 20 °C overnight. Cells were suspended in 50 mL Denature Buffer (8 M urea, 0.1 M Na 2 HPO 4 , 20 mM Tris) with pH 8.0. Recombinant AGO1-N protein was purified with a 1 mL HisTrap FF column (Cytiva) and fast protein liquid chromatography (FPLC) (AKTA purifier, GE Healthcare). The procedure was manipulated following the FPLC user manual. A sample loop (Cytiva) was used for sample binding. Denature buffer with reducing pH was used to wash the column and to elute the protein. Two mg of AGO1-N was used as antigen for rabbit immunization. A total of 10 mL of serum was applied to a HiTrap Protein A FF column (Cytiva) via FPLC. The column was washed with 20 mL wash buffer (0.5 × PBS, 0.15 M NaCl, pH 7.4). IgG was eluted with 0.1 M glycine, pH 2.5, and dialyzed in 1 × PBS buffer at 4 °C overnight.
For IgG of AGO1-N affinity purification, the HiTrap NHS-Activated HP column (Cytiva) was used in this study. Recombinant protein at a concentration of 2 mg/ mL was dialyzed in coupling buffer (0.2 M NaHCO 3 , 0.5 M NaCl, 2 M urea, pH 8.3) at 4 °C overnight. The antigen protein was coupled to NHS-activated columns following the user manual. For affinity purification, the purified IgG was passed through the antigen-coupled column in cycles with a flow rate of 0.5 mL/min for 2 h on FPLC. The column was washed with 10 mL 20 mM Tris-HCl, pH 7.5, and washed with 20 mL of a solution containing 20 mM Tris, pH 7.5 and 0.5 M NaCl. The affinity IgG was eluted with 0.1 M glycine (pH 2.5). The eluted IgG was dialyzed in 1 × PBS buffer at 4 °C overnight. IgG was diluted with 1 × PBS, and the same volume of glycerol was added to obtain the 1 mg/mL IgG of working concentration.

Identification of the T-DNA insertion in various P1/HC-Pro plants
The genomic DNA was resequenced by next-generation sequencing (NGS) with approximately 20 million paired-end read throughput (150 nt × 150 nt). The CLC Genomics Workbench was used to generate SAM files based on the alignment results of resequencing reads with Arabidopsis genome and binary vector sequences. The SAM file was further processed to identify chimeric reads (imperfectly matched reads) using T-DNA Finder (github.com/ckhuang-git/T-DNA.Finder). These chimeric reads contained a perfectly matched region and a soft-clipped sequence. The soft-clipped sequences might contain vector sequences, Arabidopsis genomic DNA (gDNA), homopolymers or miscellaneous sequences. Soft-clipped sequences shorter than 50 nt or containing more than 10% read-length homopolymers were removed by T-DNA Finder. Soft-clipped sequences were isolated from the chimeric reads, and T-DNA Finder automatically analyzed these sequences using BLASTn with the nt database. If a soft-clipped sequence belongs to Arabidopsis, it will be exported as a text file for further analysis, such as for investigating T-DNA-mediated chromosome translocation. If a soft-clipped sequence belongs to a binary vector, it will be selected by T-DNA Finder, and the alignment between the reference sequence and chimeric read sequences will be exported as a PostScript file.

AGO1-IP and in vitro RISC assay
For AGO1-IP, 15 μL IgG and 30 μL protein A beads (Cytiva, 17,152,104,011,150) were mixed and preincubated for 30 min following the user's manual. Plant tissues (0.5 g) were ground, and the tissue lysate was extracted with 1 mL AGO1-IP buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl 2 , 0.1% Nonidet P-40, and 10% glycerol, 1 × Protein inhibitor, 5 mM DTT). The tissue lysate was centrifuged at full speed, and the supernatant was mixed with IgG beads. The reaction was incubated at 4 °C for 2 h. The AGO1 precipitates were then washed four times with IP buffer and then with 1 × PBS buffer. The amount of AGO1 in the IP product was quantified based on the standard recombinant AGO1-N protein by western blotting with the α-AGO1 antibody. The IP products were further used for small RNA extraction, western blotting, or in vitro RISC assay.
cDNA fragments, including MYB33-230, MYB33 mSeed , MYB33 mCenter , CSD2, NAC1-230, ARF16-230, ARF10-230, and ARF10-191, containing miRNA target sites were amplified with the primer sets detailed in Additional file 1: Table S1 and cloned into a pGEM-T easy vector (Promega). The DNA template for in vitro transcription was amplified for transcription. RNA was synthesized using the MEGAscript T7 Transcription Kit (Invitrogen) with the DNA template and the addition of 1 μL fresh [α-P 32 ] UTP (PerkinElmer). Unincorporated UTP was removed by G50 resin (Cytiva). The RNA transcript was separated on a 6% denaturing PAGE gel in 1 × TBE buffer [29:1 acrylamide/bis (Bio-Rad), 8 M urea], and the PAGE gel was exposed to chemiluminescence film (Cytiva) to visualize the RNA. The band with a strong signal was excised and mashed in 300 μL extraction buffer (250 mM NaOAC, 1 mM EDTA) and passed through centrifuge tube filters (Costar). The supernatant was further cleaned up using the TRIzol method. RNA was diluted with 5 μL water to obtain a radioactive strength of 300 to 500 CPM. The original data of RISC assay are provided in Additional file 2, and these results have been showed in Figs. 2, 4, and 6, respectively.
The signals for full-length substrate, 5'-and 3'-cleaved fragments were detected using ImageJ. For calculation of the RISC cleavage efficiency, the product signal (5'-cleaved fragment + 3'-cleaved fragment) was divided by the total signal (full-length substrate + 5'-cleaved fragment + 3'-cleaved fragment), and the percentages were calculated. Each cleavage efficiency was normalized to the corresponding AGO1-IP signal, which was detected using ImageJ.

Immunoblotting
For the detection of AGO1 protein in tissue lysates, 20 μL supernatant was diluted with 20 μL 1 × PBS buffer, and the lysate was denatured in 40 μL 2 × protein sample buffer. For IP-AGO1 detection, 90 μL 1 × PBS buffer and 100 μL 2 × protein sample buffer were added to 10 μL IP beads. The protein samples were boiled at 100 °C for 10 min.
The protein samples were separated and analyzed by polyacrylamide-SDS-PAGE. After electrophoresis, the separated proteins were transferred to a